Methods and kits for testing the efficacy of therapeutic compounds and other therapies in a three-dimensional matrix with respect to tumorous cells and tissue

ABSTRACT

Three-dimensional physiological matrices, methods, apparatus and kits for the expedited design, testing and evaluation of oncological remedies are provided. Key aspects of the inventions include matrices, and especially gel matrices, comprising one or more physiological fibers, which are adapted and arranged to provide conditions which permit behaviors, such as the movement of cells away from the margins of samples of target tissue through the matrix, to be evaluated in a manner that produces data useful for evaluating the oncological status and characteristics of the cells. In a further key aspect, the invention permits the in vitro testing and analyses of one or more conventional, experimental or theoretical therapies with respect to specific target tissues or cells. Among such therapies are therapeutic compounds and combinations thereof, radiation therapies, combinations of therapeutic compound and radiation and numerous other possible therapies.

PRIORITY STATEMENT

Applicants hereby claim priority to U.S. Provisional Patent ApplicationSer. No. 60/813,689, filed Jun. 15, 2006, and to U.S. Provisional PatentApplication Ser. No. 60/929,122, filed Jun. 13, 2007, and entitledThree-Dimensional Physiological Matrices, Methods, Apparatus And KitsFor The Expedited Design, Evaluation, Data Organization And ReportingWith Respect To Oncological Remedies And Therapies. All the materialsand information provided in the above-identified applications are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to matrices, methods, apparatus and kitsfor the expedited design and evaluation of oncological remedies. Keyaspects of the inventions include matrices, and especially gel matrices,which are adapted and arranged to provide conditions which permitbehaviors, such as the movement of cells from a sample of target tissue,to be evaluated in a manner that produces data useful for evaluating theoncological status of the cells. In a further key aspect, the inventionpermits the in vitro testing and analyses of one or more therapies, suchas therapeutic pharmaceutical compounds, or radiological therapies, withrespect to specific target tissues and cells removed from a patient.

BACKGROUND OF THE INVENTION

Cancer is the loss of control of one or more of the regulatory systemswhich regulate the growth of cells and tissues. An uncontrolled growthof a particular tissue or cell type is a specific type of cancer. Thereare many types of cancer. In the treatment of cancer, one or moretherapeutic remedies are typically used in attempts to cure the diseaseor ameliorate its effects. Many differences exist between classes oftumors and, indeed, individual tumors of the same type. There are manypossible therapies for cancers in general. Nonetheless, most therapiesdo not work, or do not work to the extent necessary to provide thedegree of cure, remediation or desired palliative effect for a specificcancer. Because of this, it is sometimes difficult, time-consuming andexpensive to attempt to determine what therapeutic compounds may beeffective to treat a particular tumor or cancer type. Oncologists andother physicians therefore often choose a therapy based on little or nopragmatic information regarding the specific tumor. In effect, theirdeterminations of which therapy or therapies to employ arc oftenguesses. It is therefore very important to be able to more effectivelymatch a specific effective therapy to a specific tumor, and to be ableto do so in a reasonable amount of time. As a consequence of theseneeds, many technologies have attempted to address these needs, but nonehave been acceptably successful.

Various experiments have been directed toward measuring the effects ofcertain therapies on malignant cells in vitro. One of these attempts isshown in “Effects of Radiation on a Three-Dimensional Model of MalignantGlioma Invasion” International Journal of Devl. Neuroscience, Vol. 17,issue 5-6, 643-651, August 1999, (Bauman et al.). The Bauman et al.researchers used suspensions of an established cell line, the C6astrocytoma line, to show that the three-dimensional migration of known,cultured malignant cells into a collagen matrix could be observed. Themalignant glioma cells thus cultured, disrupted and processed intocloned spheroids, which were then implanted into a gel matrix, and thensubjected to one or more doses of fractionated radiation. Changes in thedistance of invasion in response to single dose and fractionatedradiation were measured over a period of 5 days.

Similar experiments were reported in “Effects Of Radiation On A Model OfMalignant Glioma Invasion”, Journal of Neuro-Oncology 44: 223-231, 1999,(Baumann et al.). In this reference, the Baumann et al. researchers usedthe same C6 cell line and experiment-al protocols to test BCNU anddexamethasone, and to compare these results with those of radiationdosing on the transformed cells.

Significantly, in both series of experiments reported by Bauman et al.,the cloned C6 astrocytoma cells were subjected to disruption bytrypsinization, and also subjected to centrifugal forces for 3-4 weeksin spinner flasks. Thus, the cells of Bauman et al were already known tobe transformed to a great extent, were generations removed from theoriginal tissue, required disruptive chemical processing, and were asubset of cloned malignant cells at the time they were subject toradiation doses. C6 astrocytoma cells were maintained in tissue cultureas cloned representatives of malignant rat glioma cells. Indeed, thecells of the Bauman et al. experiments were removed and established as acell line years before they were subjected to the experiments of Baumanet al.

In significant contrast, the present invention uses a sample quantity offresh tissue taken directly from an animal, such as a human patient.Significantly, the samples used in the present invention are notdisrupted by trypsin or other enzymes, but are mechanically divided intosample portions of appropriate sizes for testing. Thus, the presentmethods maintain the cell-to-cell contact of the sample tissue as if itwere still in vivo. An additional difference pertains to the fact thatthe experiments of Bauman et al. used an established cell line, that is,cells that were already known to have been transformed long before, andin an unknown way, to an extent great enough that they could be used toestablish a tissue culture cell line. One could not therefore expect thecells of Baumann et al. to behave in a manner reasonably replicative offresh or in vivo cells.

Others have attempted to provide ways of evaluating the response oftumor cells to chemotherapeutics. In U.S. Pat. No. 5,242,806 toYen-Maguire et al., entitled Method For Conducting The CytotoxicityAssays On Tumor Cells, a “growth matrix” of bovine cornea endotheliumcells is sometimes employed as a coating in the wells of multi-wellplates in order to facilitate the attachment of cells to the platesurfaces. Essentially, Yen-McGuire discloses ways of culturing cellularsuspensions which have been grown in two dimensions, and then assayingthe responses of the processed cells to various cytotoxic orchemotherapeutic compounds.

The cellular suspensions of Yen McGuire are provided with a defined,selective growth medium which is designed and formulated to promote thegrowth of epithelial tumor cells while inhibiting the growth of normalcells. Yen-McGuire thus teaches the use of selective nutrition to skewcellular growth and behavior. Indeed, the relative amount of cellulargrowth is measured to provide information regarding the sensitivity ofthe highly processed tumor cells. Thus, Yen-McGuire does not provide anyanalysis with respect to an original tumor sample or fragment thereof,nor does it comprehend the advantages of an in vitro system whichreplicates significant aspects of the three-dimensional environment oftumor tissue in vivo.

These problems regarding the lack of analytic tools which areeffectively usable to provide information specific to particular tumors,combined with the fact that cancer often progresses rapidly, havecreated a significant need for means and methods to quickly obtaininformation useful for testing and evaluating specific therapies, forexample, therapeutic compounds, to determine their effectiveness withrespect to particular tumors or cancer types. There is thus asignificant need for such means and methods.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to remedy theabove-mentioned drawbacks by providing three-dimensional matrices, suchas gels, as well as methods and kits for evaluating and testing tissueand cells to determine if they are abnormal, pre-cancerous, orcancerous.

Another object of the present invention is to provide methods and kitsfor testing one or more therapeutic substances to determine theirefficacy against a particular tumor or type of tumor.

In accordance with these and other objects, and in accordance with thecontext of the invention with respect to providing novel and non-obviousimprovements in all relevant fields, multiple means and methods ofpracticing the inventions are provided.

In accordance with these and other objects, means and methods forevaluating the oncological characteristics of at least one tissue orcells from an animal, such as a human being, which cells or tissue aresuspected of being abnormal, cancerous or pre-cancerous, are provided.In one preferred embodiment, a method of the invention comprises thesteps of A) obtaining a sample quantity of the suspected tumor tissue orcells from the animal, wherein the tissue comprises one or more types ofcells, B) implanting at least part of the sample of the suspected tumortissue at least partially within a three-dimensional physiologicalmatrix, the matrix being adapted and arranged for measuring one or moreparameters of the behavior of the cells of the suspected tumor tissue,C) providing sufficient nutrition to the cells of the tissue or cells sothat the parameters can be measured, D) incubating or culturing thetissue and the cells in an environment suitable for the growth of thecells and the tissue for a time sufficient to obtain measurements withrespect to one or more parameters, and E) measuring some of those one ormore parameters to obtain data regarding those behaviors.

Methods of the present invention are further adapted such that,preferably, the implantation of the sample, or portions thereof, iseffected with minimal disruption to the cells or the tissue. Forexample, in a typical procedure according to some preferred embodimentsof the invention, a sample quantity of a tissue is removed from apatient by way of a biopsy or surgery. The structural integrity of thattissue sample is then maintained to the maximum extent possible beforeits implantation. In this regard, it is noteworthy that non-disruptionof the sample means, as examples, that the sample is not homogenized, itis not chemically disrupted by strong acids, or by enzymes such astrypsin, nor is it comminuted, crushed or subjected to highconcentrations of strongly basic compounds. In sharp contrast, a sampleaccording to the invention is cut, torn or chopped into small pieces,such as chunks approximating cubes of 1.0-3.0 mm on an edge. While notintending to be bound by any underlying mechanism, the present inventorstheorize that, by minimizing the disruption to the sample tissue andcells, their respective behaviors in relation to a physiological matrixof the invention remains similar to their corresponding behaviors invivo.

In this same vein, it is preferable that implantation of the samplesduring the methods of the invention take place as soon after the sampleis removed from the animal as is reasonably possible. Thus, a removedtissue sample is preferably immediately cooled by placing it inproximity to ice, for example, and implantation is most preferablyaccomplished within one or a few hours after removal. Althoughimplantation is most preferably accomplished within an hour or so ofsample removal, the present methods provide for later implantation whencircumstances dictate as much, such as when the sample must betransported some distance to a laboratory for preparation. In any event,implantation should most preferably take place within four hours ofremoval of the sample tissue, or more preferably within 24 hours ofremoval, and less preferably within 48 hours of removal of the tissuefrom the subject animal. As one of skill in the oncological arts willappreciate, the effects of the delay in implantation and evaluation of atissue sample may depend upon many factors, including the type oftissue, its oncological status, and the conditions under which it hasbeen stored. Thus, it may be possible to delay implantation for manyhours or many days.

As another advantage of the invention, its means and methods providedata for physicians and other evaluators to make determinations orestimates regarding the oncological status of a tissue and its cells.Thus, from the data obtained, a determination can be made regardingwhether the tissue and cells are abnormal, pre-cancerous or cancerous.As a further advantage, the present means and methods permit the testingof many types of therapies, including pharmaceutical compounds andnon-chemical therapies. The present means and methods can therefore beused to provide data useful for tailoring specific individualtherapeutic regimens directed toward the precise tissue being evaluated.To this end, some preferred embodiments of the present invention furthercomprise the step of transmitting the data to at least one evaluator. Inthe context of the invention, an evaluator is any person, group ofpeople, or network of people, or any machine, computer or device adaptedand arranged for evaluating the data garnered through use of the presentmeans and methods. Evaluators include, but are not limited to the groupcomprising technicians, technicians, physicians, physician's staffmembers, physician's assistants, hospital employees, clinic employees,the patient, technologists, technical assistants, laboratory assistants,oncological analysts, nurses, nurses' assistants nurse practitioners,computers, computer-aided devices, computer-facilitated devices, andhuman or computerized agents for any of the foregoing.

In accordance with still further aspects of some preferred embodimentsof the invention, the removed sample quantity of tissue is divided intoa plurality of portions, and each of the portions is subjected to thesome or all steps of the invention. Thus, tests and evaluations of thetissue and cells can be performed in multiples, and one or moretherapies can be tested concurrently in relation to the specific sampleif desired.

Moreover, the measurements taken with respect to each portion of asample are taken over time, either at periodic intervals, or at randomintervals. In some preferred embodiments therefore, Step E is performedmore than once, or a plurality of times with respect to the separateportions of the sample. Preferably, these measurements are effectedduring the culturing or incubating of the tissue and cells as in Step Dabove.

Advantageously, means and methods of the invention can be used onvirtually any type of animal, including human medical patients, and canbe used also to evaluate tissue from other animals. Other animalsinclude, but are not limited to non-human primates, equines such ashorses and donkeys, bovines such as cows and deer, canines such as dogsand wolves, felines such as lions and domestic house cats, murines suchas mice and moles, porcines such as pigs and peccaries, avians such asbirds and penguins, amphibians such as salamanders and turtles, andreptilians such as snakes and alligators. The animals mentioned hereincomprise an exemplary listing, and not an exclusive one.

Methods of the invention also include where the measurements taken, asin Step E, are performed more than once with respect to each of theseparate portions of the sample, and wherein the measuring is effectedduring the culturing or incubating of Step D. A significant aspect ofthe invention concerns the physical relationship between the tissuesample portion and the three-dimensional matrix in which it isimplanted. Preferably, the tissue portion is disposed either partiallysubmerged in the matrix, or completely submerged therein. It isimportant that the tissue be in intimate contact with the matrix. Thus,the physiological matrix replicates to some extent the three-dimensionalenvironment in which most tissues function in vivo. Tissue samples, orportions thereof, may be implanted in any method, manner or way, whichyields the desired results

As an aid to implantation, the matrices of the invention, which aretypically gels or other permeable solids or semi-solids, can be providedin shapes which facilitate the implantation of one or more types oftissues and cells, and which facilitate the provision of nutrition tothe cells and tissue. Thus, any type of depression such as a hollow,cavity, slit, chamber or slot can be provided within the matrix itself,and can be adapted and arranged to receive the tissue or cells forimplantation, or can be adapted and arranged to hold nutrition for thecells and tissue. For example, a gel matrix according to the inventionmay comprise a hollow, cavity, slit, chamber or slot of an appropriatevolume and disposition to receive an aliquot, such as a 0.2 ml. aliquotof 10× media, as nutrition for the implanted cells and tissue. Suchdepressions can also serve as receptacles or reservoirs for drugs to betested.

In some preferred embodiments of methods of the invention, the tissue orcells are implanted in the matrix as the matrix is being formed into agel or other permeable solid or semi-solid. In these embodiments, thetissue and cells are mixed along with the components of the matrix andare thus partially or completely submerged within the matrix by the timeit gelates or hardens into a semi-solid or solid. In those preferredembodiments where the matrix is already formed, the tissue or cells canbe implanted in the matrix by physical insertion after it has formedinto a gel, solid or semi-solid.

It is important to note that solutions for forming matrices of theinvention should be essentially free of bicarbonate. By maintaining thisbicarbonate-free condition, the gels or semi-solids of the inventionadvantageously form at room temperatures and are thus capable of beingmixed and formed in a manner that is convenient, user friendly, andadaptable to many laboratory and non-laboratory environments.

In accordance with other aspects of the invention, sufficient nutritionis provided to the cells and tissue to be evaluated or tested, either asa component of the matrix as it is being formed, or as nutrition addedafter the matrix is formed, or both. The nutrition must be sufficient togrow the cells and tissue for a sufficient length of time to obtain thedesired results. In some embodiments, the sufficient or additionalnutrition preferably comprises one or more fluids, wherein the one ormore fluids are selected from the group comprising tissue culturemedias, concentrated tissue culture medias, and any culture ornutritional medias which are suitable for the tissue sample. The meansand methods of the present invention thus include wherein theconcentrated medias are one or more selected from the group comprising10× medias, and any other concentrated medias.

In another aspect of the invention, the three-dimensional physiologicalmatrix comprises the sufficient nutrition as a component of the matrixitself as it is being mixed and formed or allowed to gel. As a furtheraspect of the invention, a step of providing at least one source ofadditional nutrition to the tissue and the cells is provided. Thus, thetissues and cells may avail themselves of one or both of the sufficientnutrition and of the additional nutrition to the extent necessary sothat the parameters can be measured. In accordance with similar aspectsof the invention, the at least one source of additional nutrition maycomprise one or more of tissue culture medias, concentrated tissueculture medias, and any culture or nutritional medias which are suitablefor the tissue sample. As a further aspect of the invention, the atleast one additional source of nutrition may be supplemented with one ormore components selected from the group comprising sera, proteins,sugars, salts, lipids.

A further method of the present invention wherein the measuring of StepE is performed at periodic intervals. Preferably the measuring step isperformed at periodic intervals such as one or more intervals selectedfrom the group comprising every six hours, every 12 hours, every 18hours, every 24 hours, every 36 hours, every 48 hours, every 72 hoursand every 96 hours. Moreover other intervals are one or more selectedfrom the group comprising every day, every second day, every third day,every fourth day, and every fifth day.

As another advantage, the data obtained by the measurements of theinvention is recorded in a fixed media. Such fixed media can be any typeso long as it is capable of holding the obtained data in such form as tobe useable later. For example, paper, voice recordings, videorecordings, photographs, photomicrographs, digital recordings, any fixeddigital data means. According to the invention one or more of a humanobserver, a still camera, a video camera, an automated still camera, anautomated video camera, an infrared camera, and an automated infraredcamera can be used to obtain the desired data.

Preferably the culturing of tissues and cells according to the inventionoccurs in a period of time sufficient to obtain the desired parametersand values. For example, the periods of time during which the culturingof tissues and cells is effected includes those of at least 10 hours, atleast 36 hours, at least 72 hours and at least 125 hours, or even longerdepending on the conditions of the tissue and how the method is beingconducted

In accordance with other aspects of the invention, the sufficientnutrition is provided to the tissues and cells by placing the sufficientnutrition in contact with the matrix after the sample has been placed inthe matrix and the matrix has equilibrated to room temperature, and to asemi-solid or gel. Preferably, the room temperature is in the range offrom 10 to 30 degrees Celsius. In accordance with other aspects, thesufficient nutrition may be provided to the tissues and the cells byplacing the it in contact with the matrix after the sample has beenplaced in the matrix, and the matrix has equilibrated to a gel at roomtemperature. In other embodiments, the sufficient nutrition may beprovided to the tissues and the cells by continuous or intermittentperfusion or flow over, around, or through the matrix. In yet anotheraspect, the three dimensional quality of the matrix of the invention canbe formed itself to comprise the sufficient nutrition. The environmentfor incubating or culturing the cells and the tissue preferably includesa temperature range of from 30 to 40 degrees Celsius, a carbon dioxidetension of from 2 to 13%, and a relative humidity of from 50% to 100%.

In accordance with other aspects, methods of the invention can beconducted wherein the sample quantity is preferably divided intoportions, and each of these portions is subjected concurrently andseparately to the method, wherein the matrix comprises a gel, andwherein the gel is suitable for measuring the one or more parameters toobtain the desired values. Thus, multiple equivalent matrices may beformed in multiple containers, such as multiple containers comprising amulti-well apparatus, such that multiple portions of the sample can beevaluated concurrently. Thus, the present invention includes where aparticular tissue can be evaluated at multiple times, with respect tomultiple portions of the sample, and with respect to multiple therapies.

Advantageously, the present invention can be used to measure or obtaindata with respect to one or more parameters regarding the behavior ofthe cells and tissue. Those parameters include one or more of thedistance which individual cells migrate from the tissue sample, theaverage distance of migration of a group or population of the cells fromthe tissue sample, the distance which individual cells migrate from thetissue sample with respect to time, the average distance of migration ofa group or population of the cells from the tissue sample with respectto time, the velocity of migration of one or more designated individualthe cells per selected time period, the velocity of migration of a groupor population of designated the cells per selected time period, thenumber of migrational cells per unit area of a microscopic visual field,the speed of proliferation of the cells, the speed of unidirectionalmigration of the cells, the speed of bidirectional migration of thecells, the speed of tri-directional migration of the cells, thefrequency of directional change of the migrating cells, the number ofdirectional changes per unit time of the migrating cells, the rate ofmitosis of the migrating cells, the number of cells migrating per unittime, the number of cells in a unit area of a portion of the gel, thechange in the number of cells in a unit area of a portion of the gelwith respect to time, the number of cells in a unit volume of a portionof the gel with respect to time, and the change in the number of cellsin a unit volume of a portion of the gel, the proportion of cells whichchange migrational direction during the test period, the speed ofmigrating cells in a particular visual field, the density of migratingcells per unit area of a visual field, the migrational distance ofindividual migrating cells per unit area of a visual field, the averagemigrational distance of a group or population of migrating cells perunit area of a visual field, osmotic pressure, ionic strength, thechange in pH with respect to time, the change in pH with respect to theamount of cell migration, oxygen consumption, glucose consumption, andthe number of migrating cells in a particular direction per unit area ofa visual field.

In accordance with other advantageous aspects of the invention thethree-dimensional physiological matrices for use with the invention canbe adapted and arranged to have whatever pH value or range necessary toculture and evaluate the specific target tissues and cells. Preferably,the pH range for testing most tissues is from 5.0 to 8.0, and, morepreferably, from 7.0 to 7.5. Thus, the physiological matrix preferablyhas a pH which is adapted to a range suitable for one or more of aparticular tissue, a particular cell type or types, or to a particulartumor.

Preferably, a three dimensional matrix of the invention comprises atleast one natural or synthetic fiber, for example, one or more fibersselected from the group including type I collagens, type II collagens,type III collagens, type IV collagens, fibrin, fibrinogen, extracellularmatrix proteins derived from one or more animals, laminin, fibronectin,anti-laminin, and other natural or synthetic fibers or cables suitableto the formation of a matrix mesh or network capable of supporting cellsupport, movement and growth. In some preferred embodiments aphysiological matrix according to the invention preferably comprises agel, the gel being suitable for measuring at least one or more of theparameters necessary for evaluating the tissue, the cells, possibletherapies, and is also adapted and arranged for measuring the efficacyof one or multiple therapeutic compounds.

The means and methods of the present invention are especially adaptedand arranged to test the efficacy of virtually any therapy regimen,including pharmacologic therapeutics. In one context therefore, aphysiological matrix of the invention is adapted and arranged formeasuring the efficacy of one or more therapeutic compounds, such asknown and unknown single compounds, and known and unknown therapeuticcocktails. As examples, and not as an exclusive listing, one or moretherapeutic compounds for use or evaluation in the context of theinvention may be selected from the group comprising anti-tumor agents,DNA damaging agents such as alkylating agents, antibiotics which affectnucleic acids, platinum compounds, anti-mitotics, cell cyclestimulators, anti-metastatic agents, anti-metabolites, camptothecinderivatives, hormone therapies, biological response modifiers,interferon, anti-invasives, anti-invasion agents, anti-migration agents,anti-angiogenesis agents, and apoptotic agents, radiosensitizers,radiation, and any known, theoretical or experimental therapeuticsdirected against cell growth, cell invasion or cell viability, pro-tumoragents, pro-mitotic agents, pro-metastatic agents, pro-invasion agents,pro-migration agents, pro-angiogenesis agents, and anti-apoptoticagents.

More specifically, the three-dimensional physiological matrices of theinvention may be adapted and arranged for measuring the efficacy of oneor more therapeutic compounds, such as those selected from the groupcomprising Temozolomide (TMZ), irinotecan, Procarbazine, Methotrexate,Carboplatin, Adriamycin Cisplatin, Vincristine, Paclitaxel,1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), carmustine (BCNU),Cyclophos-phamide, Docetaxel, fluorouracil (5FU), Cytarabine,doxorubicin, bleomycin, topotecan, tamoxifen (TMX), and imatinibmesylate (Gleevec) and all other therapeutics shown to be appropriatefor treatment.

As additional examples, a physiological matrix of the invention isadaptable for measuring one or a cocktail of therapeutic compoundscomprising one or more, or at least two compounds selected from one ormore of CAF (Cyclophosphamide/Adriamycin/Fluorouracil); CMF(Cyclophosphamide/Methotrexate Fluorouracil); CMFVP(Cyclophosphamide/Methotrexate/Fluorouracil/Vincristine/Prednisone); PCV(Procarbazine/CCNU/Vincristine); ICARBO-E(Ifosfamide/Carboplatin/Etoposide); TAP (Taxol/Adriamycin/Cisplatin);EMA-CO(Etoposide/Methotrexate/Actinomycin/Cyclophosphamide/Vincristine); VBP(Vinblastine/Bleomycin/Platinol (Cisplatin)); BPD-T:(Bcnu/Platinol/Dacacarbazine/Tamoxifen); and T-10(Methotrexate/Bleomycin/Cyclophosphamide/Dactino-mycin/Adriamycin),Dactinomycin/Adriamycin) and any other actual or theoreticaltherapeutics which might be appropriate for treatment.

As yet another advantage, the means and methods of the invention may beadapted and arranged to test the efficacy of non-chemical therapies. Asexamples, and not to be taken as an exclusive listing, non-chemicaltherapies subject to being evauated or tested in the context of thepresent invention include one or more selected from the group comprisingradiation therapies, brachiotherapies, herbal therapies, naturopathictherapies, experimental therapeutic compounds, and new therapeuticcompounds.

In additional aspects, the method may further comprise the step ofdetermining the oncological status of the cells of the removed tissuefrom the data thus obtained. Preferably, the methods may furthercomprise the step of recording the data in some fixed media so that itcan be analyzed, communicated to others, and used as a record of theprocedures and their results.

In accordance with other objects of the invention, methods forevaluating the efficacy of one or more therapeutic compounds withrespect to one or more tissues comprising cells from an animal areprovided. Methods of the invention are particularly relevant withrespect to tissue and cells which are suspected of being abnormal,cancerous or pre-cancerous. Indeed, the present means and methods areespecially adaptable to testing or evaluating one or multipletherapeutic compounds with respect to specific tissue and cells. As aresult of this ability to evaluate potential therapies, such astherapeutic compounds, with respect to an exact tissue, time, effort andexpense are saved in terms of the determination of an appropriatetherapeutic regimen.

In one preferred embodiment, the present methods for evaluating one ormultiple therapeutic compounds comprises the steps of A) obtaining asample of the suspected tissue from the animal, wherein the tissuecomprises one or more types of cells, B) implanting a portion of thesample of the suspected tissue at least partially within athree-dimensional physiological matrix, the matrix being adapted andarranged for measuring one or more parameters of the behavior of thetissue and the cells, C) providing sufficient nutrition to the cells andthe tissue so that the parameters can be measured, D) providing at leastone of the one or more therapeutic compounds to the cells and thetissue, E) incubating or culturing the tissue and the cells with the oneor more therapeutic compounds in an environment suitable for the growthof the cells and the tissue, and for a time sufficient to obtainmeasurements with respect to the one or more parameters, and F)measuring one or more of the parameters to obtain data regarding thebehaviors with respect to the one or more therapeutic compounds.

Advantageously, the methods may further comprise the step of, from thedata thus obtained, determining the efficacy of the one or moretherapeutic compounds with respect to the tissue and the cells.Preferably, methods of the invention may further comprise the step ofrecording the data in some fixed form, and may further comprise the stepof transmitting the data to at least one evaluator. Evaluators and therecordation of data obtained by the present means and methods are alsodiscussed herein.

The invention is also especially adapted for circumstances wherein thesample quantity is divided into portions, and each of the portions issubjected concurrently and separately to the method, wherein the methodincludes the use of one or more controls adapted for evaluating the oneor more therapeutic compounds. Thus, the present methods can include theuse of one or more controls adapted for evaluating the one or moretherapeutic compounds with respect to the particular tissue, and withrespect to specific conditions created by the test or testing lab orclinic. Methods of the invention also include where Step D is performedduring Step E, and wherein the measuring of Step F is performed morethan once with respect to each of the separate portions of the sample,and wherein the measuring is effected during the culturing or incubatingof the tissue and cells. Preferably, the tissue portion is disposedeither partially submerged in the matrix, or completely submergedtherein.

It is important that the tissue be in intimate contact with the matrix.Thus, the physiological matrix replicates to some extent thethree-dimensional environment in which most tissues function in vivo.Tissue samples, or portions thereof, may be implanted in any method,manner or way, which yields the desired results. Means and methods ofthe invention can be used also where the implanted tissue is obtainedfrom one or more of a liquid tissue, a cell suspension, cells or groupsof cells which have been palletized, and wherein the tissue or cells areimplanted in the matrix after the matrix has formed into a gel or otherpermeable solid or semi-solid.

Means and methods of the invention can be used with virtually any animaltissue as described herein. Moreover, it is important to note thatmatrices of the invention should be essentially free of bicarbonate whenthe matrix is being formed into the gel, semi-solid or permeable solid.As an additional advantage, methods of the invention can be practicedwith respect to one or more therapeutic compounds, for example where asingle compound is tested and the method is performed with at least oneset of controls. As one skill in the art of testing oncological remedieswill appreciate, any number of controls, therapeutic substances,therapeutic methods, experimental compounds, or other cancer treatmentssuch as radiation, radiotherapy or any other actual, theoretical orexperimental therapy of method can be tested with the present inventionin in vitro conditions.

Moreover, therapeutic compounds can be provided to the tissues and cellsas a component of the sufficient nutrition. More therapeutic compoundsare provided to the cells and the tissue as a component of thephysiological matrix. As a component after the sufficient nutrition isprovided to the cells and tissues or wherein the one or more therapeuticcompounds include two compounds and the method is performed with atleast one set of controls. It is similarly possible to use the presentinvention and methods to test three or more therapeutic compoundspreferably with respect to at least one set of controls. Methods ofphysiological matrix is adapted and arranged for measuring the efficacyof one or more therapeutic compounds, and wherein the one or moretherapeutic compounds are selected from the group comprising DNAdamaging agents such as alkylating agents, antibiotics which affectnucleic acids, platinum compounds, anti-mitotics, cell cyclestimulators, anti-metastatics, anti-metabolites, camptothecinderivatives, hormone therapies, biological response modifiers,interferon, anti-invasives, radiosensitizers, radiation, and anyestablished, theoretical or experimental therapeutics directed againstcell growth or cell invasion.

The present means and methods with respect to a three-dimensionalphysiological matrix of the invention are adapted and arranged formeasuring the efficacy of one or more therapeutic compounds.

More specifically, a physiological matrix of the invention is adaptedand arranged for measuring the efficacy of one or more therapeuticcompounds comprises at least two therapeutic compounds. And where atleast two therapeutic compounds are selected from one or more of

It is again necessary to mention that the physiological matricesaccording to the invention should be essentially free of bicarbonate,for example wherein the matrix is formed from components that areessentially free of bicarbonate, and the final gel form of the matrix isthus essentially bicarbonate free.

As another distinct aspect of the present methods one or more parameterscan be obtained regarding the behaviors of one or more therapeuticcompounds, and the behaviors of the cells themselves. Parameters thusmeasured yield data. In turn, such data can be used to evaluate theperformance of the cells, or the performance of one or moretherapeutics, or the performance of one or more other types of therapieswith respect to the tissues and cells tested. Preferably, the measuringstep E is performed at periodic intervals, and wherein the periodicintervals are appropriate to one or more of the cells, the tissue andthe therapy or therapies being tested or evaluated.

Preferably the measuring of Step E is performed at periodic intervalsfor a period of time of between one hour and 15 days. More preferably,the periodic intervals are one or more selected from the groupcomprising every six hours, every 12 hours, every 18 hours, every 24hours, every 36 hours, every 48 hours, every 72 hours and every 96hours. Moreover other intervals such as every day, every second day andevery third day, every fourth day, every fifth, day every sixth day,every seventh day, every eighth day, and every ninth day. Methods of thepresent invention wherein the measuring of Step E is performed atnon-periodic intervals, and wherein the non-periodic intervals areappropriate to one or more of the cells, the tissue and the therapy ortherapies being tested or evaluated.

As another advantage, the data obtained by the measurements of theinvention is recorded in a fixed communications media. Such fixed mediacan be any type so long as it is capable of holding the obtained data insuch form as to be useable later. For example paper, voice recordings,video recordings, photographs, photomicrographs, digital recordings, anyfixed digital data means, any computer network-facilitated means,infrared recording, ultrasound recordings, and magnetic resonancerecordings. According to the invention one or more of a human observer,a still camera, a video camera, an automated still camera, an automatedvideo camera, an infrared camera, and an automated infrared camera.

Preferably the culturing and/or incubating of tissues and cellsaccording to the invention occurs for a period of time sufficient toobtain data regarding the desired parameters and values. For most tissueand cells to be tested, that period of time is between one hour and 15days. More specifically, the culturing or incubating of the tissue andthe cells typically occurs for a period of time of at least 1 hour, orat least 10 hours, or at least 24 hours, or at least 36 hours or atleast 72 hours, or at least 125 hours, or at least 175 hours, or atleast 200 hours, or at least 250 hours or at least 300 hours.

In some preferred embodiments, the environment for incubating orculturing the cells and the tissue includes a temperature range of from26 to 43 degrees Celsius, a carbon dioxide tension of from 2.0% to13.0%, and a humidity range of from 50-100%.

In accordance with other aspects of the invention, the sufficientnutrition comprises one or more fluids, and wherein the one or morefluids are selected from the group comprising tissue culture medias,concentrated tissue culture medias, and any culture or nutritionalmedias which are suitable for the tissue sample Preferably theconcentrated medias are one or more selected from the group comprising10× medias, and any other concentrated medias. Preferably, thesufficient nutrition is provided to the tissues and cells by placing thesufficient nutrition in contact with the matrix after the sample hasbeen placed in the matrix

The method of the invention further comprises the step of J) providingat least one source of additional nutrition to the tissue and the cellsso that the parameters can be measured. Wherein at least one source ofadditional nutrition comprises one or more of tissue culture medias,concentrated tissue culture medias, and any culture or nutritionalmedias which are suitable for the tissue sample and wherein least oneadditional source of nutrition is supplemented with one or more selectedfrom the group comprising sera, proteins, sugars, salts, lipids.Preferably the sufficient nutrition is provided to tissues and cells byplacing the sufficient nutrition in contact with the matrix after thesample has been placed in the matrix and the matrix has equilibrated toroom temperature, wherein the room temperature is in the range of from10 to 30 degrees Celsius.

Advantageously, the present invention can be used to obtain dataregarding one or multiple parameters of the behaviors of the tissue andcells being evaluated or tested as discussed herein. Significant amongthose parameters are the distance which individual cells migrate fromthe margins of the tissue sample, the average distance of migration of agroup or population of the cells from the margins of the tissue sample,the distance which individual cells migrate from the tissue sample withrespect to time, and the number of migrational cells per unit area of amicroscopic visual field, that is, cell density in a portion of the gel.

As an additional advantage, methods of the invention may include thosefor evaluating the efficacy of one or more therapies with respect to atleast one tissue from an animal, the tissue comprising cells, and thetissue being suspected of being abnormal, pre-cancerous or cancerous,the method comprising the steps of A) obtaining a sample of the tissuefrom the animal, wherein the tissue comprises one or more types ofcells, B) implanting the sample at least partially within athree-dimensional physiological matrix, the matrix being adapted andarranged for measuring one or more parameters of the behavior of thecells of the tissue or cells, C) providing sufficient nutrition to thecells of the tissue of the portions so that the parameters can bemeasured, D) subjecting the cells and the tissue to at least one of theone or more therapies, E) incubating or culturing the tissue and thecells of the portions in an environment suitable for the growth of thecells and the tissue for a time sufficient to obtain measurements withrespect to the one or more parameters for each of the portions, and withthe one or more therapeutic compounds, F) measuring, with respect toeach of the portions, one or more of the parameters to obtain dataregarding the behaviors with respect to the one or more therapies.

In accordance with these and other objects of the invention, athree-dimensional physiological matrix suitable for culturing at leastone tissue from an animal, the tissue comprising cells, is provided. Inone aspect, the tissue or cells of the sample are suspected of beingabnormal, cancerous or pre-cancerous. In another, the physiologicalmatrix comprises A), at least one natural or synthetic fiber means,wherein the fiber means is adapted and arranged such that the cells, ifabnormal, cancerous or pre-cancerous, are enabled to grow away from thesample into the matrix when the sample is implanted at least partiallyin the matrix, B), a sufficient amount of at least one additivecomprising sufficient hydroxyl or hydrogen groups to bring the effectivepH range of the matrix into an acceptable range, and C), sufficientnutrition to sustain the cells and the tissue in the matrix. It isimportant to note that the matrices of the invention and the sufficientnutrition should be essentially free of bicarbonate when they are beingformed.

The three-dimensional physiological matrix is mixed from components andformed into a solid, a semi-solid, or a gel, and the matrix is adaptedand arranged to permit migration of the cells away from the margins ofthe tissue and into the matrix. Moreover, the matrix is formulated,adapted and arranged to accept the sample as soon as the solid or gel isformed, or during its formation.

Preferably, a three-dimensional physiological matrices of the inventioncomprise at least one natural or synthetic fiber, for example, one ormore selected from the group comprising type I collagens, type IIcollagens, type III collagens, type IV collagens, fibrin, fibrinogen,laminin, anti-lamimin, positively charged poly(L-lysine) amino acidchains, positively charged poly(D-lysine) amino acid chains, andextracellular matrix proteins derived from one or more animals. In onepreferred embodiment, the matrix comprises at least 90% Type I collagen.In another preferred embodiment of a matrix according to the invention,the gel comprises at least 95% Type I collagen.

The pH of a three-dimensional physiological matrix of the invention isbrought into an acceptable range by means of one or more additivespreferably selected from the group comprising NaOH, KOH and otherstrongly basic compounds. A matrix of the invention may further compriseadditional nutrition to be added after the formation of the matrix intoa solid, semi-solid or gel.

In accordance with other aspects of the invention, the sufficientnutrition comprises one or more fluids, preferably selected from thegroup comprising tissue culture media, concentrated tissue culture mediaor any culture or nutritional media which are suitable for the tissuesample to be evaluated. Suitable concentrated medias include one or moreselected from the group comprising 10× media, and other concentratedmedia. Preferably, the sufficient nutrition is provided to the tissuesand cells by placing the sufficient nutrition in contact with the matrixafter the sample has been placed in the matrix and wherein oneadditional source of nutrition is supplemented with one or more selectedfrom the group comprising sera, proteins, sugars, salts, lipids, and thematrix has equilibrated to room temperature, wherein room temperature isin the range of from 10 to 30 degrees Celsius.

In accordance with other advantageous aspects, a three-dimensionalphysiological matrix for use in the context of the invention can beadapted or arranged to have whatever pH value or pH ranges necessary toculture and evaluate the specific tissues and cells. Preferably, the pHrange for testing most tissues is from 5.0 to 8.3, more preferably 6.4to 7.9 and even more preferably from 7.4 to 7.6. Preferably, the tissueto be evaluated or tested is implanted or disposed completely submergedwithin the three-dimensional matrix.

In accordance with other aspects of the three-dimensional physiologicalnature of the invention, a matrix is preferably adapted and arranged tobe in sufficient proximity to at least one additional source ofnutrition, wherein the matrix is permeable to the additional source ofnutrition to the extent necessary that the sample tissue and cellsreceive nutrition sufficient to enable them to grow into the matrix tothe extent necessary to measure the one or more parameters. Moreover,the at least one additional source of nutrition may preferably includeone or more additives or supplements selected from the group comprisingserums, proteins, sugars, salts and lipids or any culture media that issuitable for the cell or tissue type. Preferably, the density of thefiber in a formed matrix of the invention is between 1.3 and 3.3 mgs/ml,more preferably from 2.0 to 2.8 mgs/ml, and even more preferably between2.3 and 2.5 mgs/ml.

Advantageously, the present invention can be adapted and arranged to besuitable for measuring at least one parameter of the behavior of cellsfrom the tissue, and at least one parameter relating to the suspectedcancerous or abnormal status of the cells or the tissue. Theseparameters are listed hereinbefore.

In a significant aspect, a three-dimensional physiological matrix of theinvention includes at least one natural or synthetic fiber selected fromthe group comprising Type I collagen, wherein the Type I collagencomprises at least 70% of the total fiber of the formed matrix, or atleast 80% of the total fiber, at least 90% of the total fiber and atleast 95% of the total fiber. In one preferred embodiment, athree-dimensional physiological matrix of the invention comprises atleast one natural or synthetic fiber means of primarily Type I collagen,and at least one additive sufficient to bring the pH of the matrix intoan acceptable range, such as a sufficient amount of 0.1 N NaOH, andwherein the sufficient nutrition is a bicarbonate-free 10× media.

In another preferred embodiment, a three-dimensional physiologicalmatrix of the invention comprises eight parts by volume of the naturalor synthetic fiber, the at least one additive comprises one part byvolume of the 0.i N NaOH, and the sufficient nutrition comprises onepart by volume of the bicarbonate-free 10× media.

In another aspect, a physiological matrix according to the invention maycomprise at least one first hollow, cavity, slit or chamber, and thefirst hollow, cavity slit or chamber may be adapted and arranged toreceive one or more tissues samples, wherein the one or more tissuessamples are in the form of one or more of a solid, a liquid, asuspension, and combinations thereof. Moreover, a three-dimensionalmatrix of the invention may comprises at least one second hollow cavity,slit or chamber, wherein each of the second hollows, cavities, slits orchambers is adapted and arranged to receive at least a portion of theadditional source of nutrition.

Preferably, the three-dimensional matrix of the invention matrixcomprises a gel, the gel being suitable for measuring at least one ofthe one or more parameters and of producing resulting data. Moreover,the physiological matrices of the present invention are adapted andarranged for measuring the efficacy of one or a plurality of therapeuticcompounds, many of which are exemplified herein. Moreover, means ormethods of the invention can be adapted and arranged to test theefficacy of non-synthetic chemical therapies, wherein the non-syntheticchemical therapies are one or more selected from the group comprisingradiation therapies, brachiotherapies, herbal therapies, naturopathictherapies, experimental therapeutic compounds, and new therapeuticcompounds, hyperthermal therapies, hyperbaric therapies, hypobaric andphotosensitizing therapies.

An additional benefit of the present invention pertains to itsamenability to kit form. Thus, in some embodiments, the invention mayinclude a kit for testing the efficacy of therapeutic compounds on anexpedited basis, the kit comprising a means for collecting at least onetissue comprising cells from an animal, the tissue being suspected ofbeing abnormal, cancerous or pre-cancerous, at least onethree-dimensional physiological matrix for culturing the tissue samplein three dimensions for a sufficient length of time to obtainmeasurements of at least one parameter of the behavior of the cells andthe tissue, a means for measuring the parameters to determine a set ofpossible therapeutic compounds which might be efficacious for treatingthe tumor or abnormal tissue, and a means for testing the therapeuticeffect of the determined therapeutic compounds.

Preferably, the behavior of cells at multiple sites, for example, atleast 2 sites or at least 3 sites, or at least 4 sites, or at least 5sites is measured to obtain data regarding the behavior of the tissue orcells. In some preferred embodiments, readings made using microscopy andvideo-microscopy include the actions or steps of measuring directly themigratory distance, speed, cell density and direction of movement of thecells or tissue with respect to the matrix upon which they are placed orcultured.

In other preferred embodiments of the invention, the suspect tissue andmatrix are then processed and treated so that they can be snap frozen,paraffin embedded, sectioned, and then assessed with respect to one ormore parameters, including, but not limited to, mitotic index/celldivision, cell growth, necrosis, apoptosis, anoikis, adhesion, cellularsignaling and invasion-related protein expression. Any number of proteinand genetic parameters can be analyzed with respect to their usefulnessin determining the oncologic state of the cells or tissues.

In accordance with certain other aspects of the invention, the matrixmay be of any configuration or material which permits the evaluation ofthe effect of therapeutic compounds on the cells or tissues. In someembodiments of the invention, the matrix preferably comprises a collagengel, the gel being suitable for measuring the one or more parameters.Matrices could include any protein or glycoprotein or sugar componentsof extracellular matrix, for example, including but not limited to,fibronectin, laminin, hylaronin, growth factors and integrins.

In general, in one set of preferred embodiments, the typical matrixelement of the invention comprises a gel. In one set of especiallypreferred embodiments, a matrix of the invention comprises protein inits native, non-denatured state, in the amount of 1.0 to 2.1 mg/ml. Onepreferred protein is, for example, Type I Collagen. Other proteins areamenable to use or adaptation as components of matrices of theinvention. In some embodiments, a matrix of the invention possesses afinal pH preferably in the range of from 6.0 to 8.5, more preferably inthe range of from 6.5 to 8.0, and most preferably in the pH range offrom 6.8 to 7.5

In other embodiments, a matrix of the invention is incubated and used inan air atmosphere having carbon dioxide preferably in the range of 1.0to 18.0%, more preferably in the range of from 2.0 to 14.0%, and mostpreferably in the range of from 4.0 to 9.0%. A carbon dioxide incubatoris useful for incubation processes according to the invention. Inaccordance with other aspects of the invention, the parameters of matrixcomposition, carbon dioxide tension, and other conditions vary accordingto tumor type. For example, for most categories of tumors, temperaturesfor incubation of the matrices combined with tissue or cells aregenerally kept within approximate physiological ranges, preferably offrom 30-44 degrees Celsius, more preferably of from 33-41 degreesCelsius, and most preferably of from 35-39 degrees Celsius.

In another advantageous aspect of some embodiments of the invention, amatrix may comprise one or more constituents provided to adapt thematrix for use with a particular tumor, or a particular class of tumors.Possible constituents to a matrix according to the invention, forexample a gel matrix, may include growth factors such as VEGF or HGF,which tends to alter the invasion patterns of the tissue cells into thematrix and enhances cell growth. Other possible constituents includestructural proteins, for instance, extracellular matrix proteins likelaminin, fibronectin, and the various collagens, for example CollagenIV.

Immunoglobulins may also be included or added to a matrix of theinvention in order to stabilize it in various respects. For instance,anti-laminin may be included in the matrix. As additional means andmethods for adapting matrices of the invention to uses with respect tospecific tumors or classes of tumors, lipids, such as human or bovinebrain extract, solids like human dura, and plant extracts such asphytostimulants and inhibitors may also be included in a matrix of theinvention in order to vary its characteristics and adapt it to specificuse. In some embodiments, other living cells may be included. Exemplarycells include melanoma cells, fibroblasts and endothelial cells

Matrices of the invention may include inert additives also. As examples,glass or plastic shapes, such as cylinders may be provided within tissueculture wells to act as structural forms for the gel so that the matrixgel can be molded to create a tiny well or indentation useful for addingcell suspensions such as leukemia or lymphoma cells to the matrix in apre-determined area in the matrix gels.

In accordance with preferred aspects of the invention, the one or moreparameters to be measured comprise one or more of: the speed ofproliferation of the cells, the speed of unidirectional migration of thecells, the speed of bi-directional migration of the cells, the speed oftri-directional migration of the cells, the rate of mitosis of thecells, the rate of change of migrational direction of the cells, theproportion of cells which change migrational direction during the testperiod, the speed of migrating cells in a particular visual field, thedensity of migrating cells per microscopic visual field, the migrationaldistance of individual migrating cells per microscopic visual field, theaverage migration-al distance of a group or population of migratingcells per microscopic visual field, and the number of migrating cellsper microscopic visual field direction.

With respect to the invention, the “Test Period” is that period of timeduring which the one or more tissues or cells are cultured and testedwith respect to their oncological behaviors, and the “EvaluationalPeriod” is that period of time during which the one or more therapeuticcompounds are evaluated with respect to efficacy in treating thespecific tissue samples or cells, and other parameters.

In accordance with the many advantageous aspects of the methods of theinvention, the one or more therapeutic compounds or treatments areselected from the group comprising, but not limited to, DNA damagingagents, anti-mitotics, cell cycle stimulators, anti-metastatics,anti-invasives, radiosensitizers, radiation, and any established,theoretical or experimental therapeutics against cell growth and cellinvasion.

Methods of the invention include the evaluation of one or moretherapeutic compounds, individually, combined with one another, as oneor more cocktails, or in any sequence, in order to determine theirlikely efficacy against the suspected tumor tissue and cells. Thus, insome embodiments, the methods of the invention include testing onetherapeutic compound, or a pair of therapeutic compounds, or threetherapeutic compounds, or four therapeutic compounds, or more than fourtherapeutic compounds, or any number of compounds in a cocktail ofcompounds, or provided at different times or sequences to the tissuebeing evaluated.

Preferably, the culturing of the tissue and the cells during the testand evaluational periods is for a length of time sufficient to obtainthe values required to make a determination of the efficaciousness ofone or more potential therapeutic compounds. The culturing of the tissueand the cells occurs for a period sufficiently long enough to obtain theneeded data. Thus, the culturing of the tissue and the cells occursduring the one or more test and evaluational periods, and thus during aperiod of time of preferably less than 10 days, more preferably of lessthan 8 days, even more preferably of less than 6 days, and mostpreferably of less than 4 days. Thus, all readings typically occurwithin 10 days from the time of removal of the tissue or cells from themammal. Basic microscopic readings of distance, direction and densityare taken manually or by automated systems, preferably at predeterminedintervals depending on the nature of the suspected tumor, the nature andamount of the tissue and the nature and number of cells or cell typesbeing evaluated. In accordance with the various embodiments of theinvention, the predetermined interval is one or more hours, for example,every 4 hours, or every 8 hours or every 16 hours or every 24 hours.

Video monitoring of the tissues and cells may also be provided in orderto gather and record additional information. The video monitoring ispreferably provided on a continuous basis at intervals. The intervalsare selected based upon the nature of the tissue and cells, the natureof the suspected tumor or tumors, and the estimation of likely behaviorof the tissue and cells on a matrix. In some preferred embodiments ofthe invention, video monitoring is provided on a continuous basis, at30-second intervals, or at 60-second intervals or at 90-second intervalsor at 120-second intervals, or at 150-second intervals.

Methods of the present invention include also those of testing theefficacy of therapeutic compounds on an expedited basis, testing theefficacy of therapeutic compounds with respect to a specific individualmammal, such as a human, and testing the synergistic efficacy of two ormore therapeutic compounds on an expedited basis.

The present invention includes also one or more kits for testing thesynergistic efficacy of therapeutic compounds on an expedited basis, thekit comprising i) means for collecting at least one tissue comprisingcells from a mammal, the tissue being suspected of being transformedinto cancerous or pre-cancerous tissue, ii) means for culturing thetissue comprising cells for a sufficient length of time to obtaindesired values, iii) means for evaluating the values to determine a setof possible therapeutic compounds which might be efficacious fortreating the tumor, and iv) means for testing the therapeutic effect ofthe determined therapeutic compounds.

As one of skill in the oncology arts will appreciate, numerousvariations of the means and methods disclosed herein fall within thescope of the present application. Moreover, the examples provided hereinare provided as inclusive illustrations of the invention, and not asexclusive limitations.

DESCRIPTIONS OF THE FIGURES

FIGS. 1-8 are photographic and photomicrographic images which show, byway of example, use of the means and methods of the present inventionwith respect to a tissue sample removed from a patient. With respect toFIGS. 1-8, Patient Sample GS-640, comprising kidney tissue, was removedfrom the patient by a surgeon during surgery.

Tissue analysis and screening in accordance with the invention wererequested by the surgeon, and proper consent was obtained from thepatient prior to surgical removal of a sample quantity of tissue fromthe RHS Kidney. The patient was diagnosed with renal cell carcinoma. Atissue sample of approximately 1 gram in quantity was removed from thepatient during surgery, and was placed into a 25 ml sample jar, andimmersed in sterile saline. The tumor tissue was kept at 4 degreescentigrade until processed for the screening of therapeutic compounds inaccordance with the means and methods of the invention. All procedureswere performed under sterile conditions in a bio-safety laminar flowhood.

FIG. 1 is a photographic image showing the mechanical division of thesample into portions to be tested. The sample has been prepared bydissecting away any non viable tissue and blood prior to mincing into1-2 mm pieces using sterile disposable scalpels.

FIG. 2 is a photographic image showing tumor sample portions which havebeen placed into each well of a 48 well plate, and been submersed in agel matrix mixture according to the invention. After insertion of theportions, the mixture was allowed to form into a gel at room temperaturefor approximately 30 minutes. Once the gel formed around the fullyimmersed tumor fragments, nutrient rich media was added as a liquidoverlay that permeated the entire gel and tumor fragment.

Each different chemotherapy and or dose of chemotherapy was added to aset of 6 wells out of the 48-well plate. A total of 7 differentchemotherapies were assessed in this plate. One set of six wells werenot treated with drug and thus acted as ‘control conditions’ for tumorfragment growth and invasion. Each of the eight vertical lanescontaining six separate wells was marked with a symbol for eachchemotherapy/dose used and noted correspondingly on data collectionsheets. FIG. 2 shows plate after chemotherapeutics and growth media havebeen added. The plates were then placed in a conventional 37 degreeCelsius incubator at 5% CO₂.

FIG. 3 is a photomicrographic image which shows the tumor sample at Day0 where the initial appearance of the tissue fragment in the lower rightof the photomicrograph shows no cell migration or invasion of cells awayfrom the margins of the sample portion and out into the gel. This imageis thus representative of all wells at Day 0 with or withoutchemotherapeutics. Images were made with a 400× magnification phasecontrast micrograph using ZEISS camera specs.

FIG. 4 is a photomicrographic image which showing a patient controlsample (no treatment) after 3 days incubation @37° C. and 5% CO₂.Noteworthy is the migration of cells away from the solid tumor mass in ahalo around the tumour fragment.

FIG. 5 is a photomicrographic image which shows a patient control sampleafter 5 days. Noteworthy is the halo of cells and increased cell densityas compared with the results shown at Day 3 of the test. The lines onthe images have been drawn to show the distance measurements withinwhich approximately 95% of the migrating cells have migrated away fromthe margins of the tissue sample.

FIG. 5 also shows the presence of fibroblasts which sometimes appear atthe later time points. Fibroblasts are noticeably different in numbersand appearance than migrating tumor cells. For example, fibroblastsappear in the surrounding gel matrix as much larger cells than themigrating tumor cells, and with significantly larger cytoplasm, as wellas the classic elongated spindle fibroblast appearance. Fibroblasts alsostain positively upon immuno-histochemical analysis for antibodies tofibroblast markers such as fibroblast growth factor. Thus, fibroblastsmay be differentiated from tumor cells in a straightforward manner.

FIG. 6 is a photomicrographic image demonstrating the effect oftreatment with Taxol™. The image of FIG. 6 shows a sample well at 5 daysafter treatment with Taxol™. Noteworthy is the change in behavior of thecells exposed to Taxol™. Specifically, a drop in both the distance andthe number of cells that have grown and invaded away from the solidtumor fragment is seen on the lower left hand side of the image.

FIG. 7 is a photomicrographic image which demonstrates the effect oftreatment with Docetaxol™. The image of FIG. 7 shows a sample wellwherein the tumor tissue has been treated for 5 days with Docetaxol™while showing a moderate response to the drug. This moderate response isevidenced by the fewer number of invading cells than the control butthose treated with Docetaxol™ have a longer migratory distance thantreatment with Taxol™.

FIG. 8 is a photomicrographic image which shows the carcinoma patientsample treated for 5 Days with Cisplatin™. This image thus shows anexample of a failed chemotherapy, being identical in appearance and cellnumbers to the control sample (FIG. 5) at the same 5 day time point.

BRIEF DESCRIPTION OF THE TABLE

The present invention is adaptable to be used with many possibletherapeutic substances, and combinations thereof, including those shownin the accompanying Table Of Exemplary Therapeutic Compounds. Inaccordance with important aspects of the invention, the attached Tableprovides only an exemplary list of therapeutic compounds which may beevaluated with the present methods and means. Virtually any othercompound or combination of compounds with potential therapeutic valuemay be tested and evaluated with the present invention. Moreover, themeans and methods of the present invention are adaptable and useful fortesting any compound or combination of compounds with respect to theirpotential therapeutic value, regardless of whether those compounds havebeen involved in the drug approval process. Thus, the present inventionprovides means and methods for initially testing and evaluatingcompounds with respect to possible efficacy against many tumors andtumor types without the necessity of using animal or human subjects.

As those of skill in the art will appreciate, numerous permutations ofthe matrices, methods, apparatus and kits of the invention are possiblewithin the metes and bounds of the information disclosed herein. Thus,although the present invention has been described with reference to someof the preferred embodiments, variations and modifications of steps,elements and components of the invention can be substituted therefore,while remaining within the spirit and scope of the invention.

TABLE OF EXEMPLARY THERAPEUTIC COMPOUNDS Quantitiy Compound 3 TMZ 250 mgcapsules (Scering) 1 Irinotecan 20 mg/ml solution × 5 ml vial(Pharmacia) 1 Procarbazine 50 mg capsule (Sigma- Tau) 1 Methotrexate 25mg/ml solution × 20 ml vial (Mayne) 1 Carboplatin 10 mg/ml solution × 45ml vial (Mayne) 1 Adriamycin 2 mg/ml solution × 100 ml vial (Novo) 1Cisplatin 1 mg/ml solution × 100 ml vial (Mayne) 1 Vincristine 1 mg/mlsolution × 5 ml vial (Mayne) 1 Paclitaxel 6 mg/ml solution × 50 ml vial(BMS) 3 CCNU 100 mg capsules (BMS) 1 BCNU powder for injection × 100 mgvial (BMS) 1 Cyclophosphamide powder for injection × 2 gm vial (BMS) 1Docetaxel 40 mg/ml solution with diluent × 2 ml vial (Aventis) 1 5FU50mg/ml solution × 100 ml vial (Mayne) 1 Cytarabine 100 mg/ml soltuion ×10 ml vial (Mayne) 1 Cytarabine 100 mg powder for injection (Pfizer)

1. A method for evaluating the efficacy of one or more therapeuticcompounds or candidate therapeutic compounds with respect to at leastone tissue from an animal, said tissue being suspected of beingabnormal, cancerous or pre-cancerous, the method comprising the stepsof: (A) obtaining a sample of said suspected tissue from said animal,wherein said tissue is dissociated mechanically by dividing the tissueinto tissue sample portions and not dissociated by chemical or enzymaticmeans, wherein said tissue comprises one or more types of cells, (B)implanting each of said tissue sample portions within athree-dimensional physiological matrix, wherein each of said portions isessentially completely submerged within said matrix, said matrix beingadapted and arranged for measuring one or more parameters of thebehavior of said tissue portion and said cells, wherein thephysiological matrix comprises at least one collagen fiber componentadapted and arranged such that the cells, if abnormal, cancerous orpre-cancerous, are enabled to grow away from the sample portion into thematrix, and wherein said collagen fiber component comprises at least onenatural or synthetic collage fiber and is present in a density of from1.3 mg/ml to 3.3 mg/ml, (C) providing nutrition to said tissue sampleportions and said cells so that parameters can be measured, (D)providing at least one of said one or more therapeutic compounds orcandidate therapeutic compounds to said tissue sample portions and saidcells, (E) incubating said tissue sample portions and said cells underapproximate physiological conditions with said one or more therapeuticcompounds or candidate therapeutic compounds for a period sufficient toobtain measurements with respect to said one or more parameters, and (F)measuring one or more of said parameters to obtain data regarding thebehavior of said tissue sample portions and said cells with respect tosaid one or more therapeutic compounds, wherein each of said portions issubjected concurrently and separately to said method, and wherein themethod includes the use of one or more controls adapted for evaluatingsaid one or more therapeutic compounds.
 2. The method of claim 1,further comprising the step of G) from said data, determining theefficacy of said one or more therapeutic compounds with respect to saidtissue and said cells.
 3. The method of claim 1, further comprising thestep of H) recording said data in some form.
 4. The method of claim 1,further comprising the step of I) transmitting said data to at least onemedical evaluator.
 5. The method of claim 4, wherein said evaluator iscomputerized.
 6. The method of claim 1, wherein said Step D is performedduring said Step E.
 7. The method of claim 1, wherein said measuring ofStep F is performed more than once with respect to each of said separateportions of said sample, and wherein said measuring is effected duringsaid incubating of Step E.
 8. The method of claim 1, wherein said tissuesample portion is implanted in said matrix as said matrix is beingformed.
 9. The method of claim 1, wherein said implanted tissue sampleportion is a liquid tissue.
 10. The method of claim 1, wherein saidtissue sample portion is implanted in said matrix after said matrix hasformed into a gel or other permeable solid.
 11. The method of claim 1,wherein said animal is one or more selected from the groups consistingof primates, equines, bovines, canids, mammals, felines, canines,murines and porcines.
 12. The method of claim 1, wherein said one ormore therapeutic compounds or candidate therapeutic compounds is asingle compound incubated with each tissue sample portion, and saidmethod is performed with at least one set of control portions, whereinsaid set of control portions is incubated free of said one or moretherapeutic compounds or candidate therapeutic compounds.
 13. The methodof claim 1, wherein said one or more therapeutic compounds or candidatetherapeutic compounds are provided as a component of said nutrition. 14.The method of claim 1, wherein said one or more therapeutic compounds orcandidate therapeutic compounds are provided as a component of saidphysiological matrix.
 15. The method of claim 1, wherein one or moretherapeutic compounds or candidate therapeutic compounds is providedafter step C).
 16. The method of claim 1, wherein said one or moretherapeutic compounds or candidate therapeutic compounds consists of twocompounds incubated with each portion of said tissue, and said method isperformed with at least one set of control portions, wherein said set ofcontrol portions is incubated free of said one or more therapeuticcompounds or candidate therapeutic compounds.
 17. The method of claim 1,wherein said one or more therapeutic compounds or candidate therapeuticcompounds is three or more compounds, and wherein said method isperformed with at least one set of control portions, wherein said set ofcontrol portions is incubated free of said one or more therapeuticcompounds or candidate therapeutic compounds.
 18. The method of claim 1,wherein said physiological matrix is adapted and arranged for measuringthe efficacy of one or more therapeutic compounds, and wherein said oneor more therapeutic compounds are selected from the group consisting ofDNA damaging agents such as alkylating agents, antibiotics which affectnucleic acids, platinum compounds, anti-mitotics, cell cyclestimulators, anti-metastatics, anti-metabolites, camptothecinderivatives, hormone therapies, biological response modifiers,interferon, anti-invasives, radiosensitizers, radiation, and candidatetherapeutics directed against cell growth or cell invasion.
 19. Themethod of claim 1, wherein said physiological matrix is adapted andarranged for measuring the efficacy of one or more therapeuticcompounds, and wherein said one or more candidate therapeutic compoundsare selected from the group consisting of Temozolomide (TMZ),Irinotecan, Procarbazine, Methotrexate, Carboplatin, AdriamycinCisplatin, Vincristine, Paclitaxel,1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), carmustine (BCNU),Cyclophosphamide, Docetaxel, fluorouracil (5FU), Cytarabine,doxorubicin, bleomycin, topotecan, tamoxifen (TMX), and imatinibmesylate (Gleevec).
 20. The method of claim 1, wherein said at least twotherapeutic compounds are selected from one or more of CAF(Cyclophosphamide/Adriamycin/Fluorouracil); CMF(Cyclophosphamide/Methotrexate Fluorouracil); CMFVP(Cyclophosphamide/Methotrexate/Fluorouracil/Vincristine/Prednisone); PCV(Procarbazine/CCNU/Vincristine); ICARBO-E(Ifosfamide/Carboplatin/Etoposide); TAP (Taxol/Adriamycin/Cisplatin);EMA-CO(Etoposide/Methotrexate/Actinomycin/Cyclophosphamide/Vincristine); VBP(Vinblastine/Bleomycin/Platinol (Cisplatin)); BPD-T:(Bcnu/Platinol/Dacacarbazine/Tamoxifen); and T-10(Methotrexate/Bleomycin/Cyclophosphamide/Dactinomycin/Adriamycin). 21.The method of claim 1, wherein said method is adapted and arranged totest the efficacy of non-synthetic chemical therapies.
 22. The method ofclaim 1, wherein said non-synthetic chemical therapies are one or moreselected from the group consisting of radiation therapies,brachiotherapies, herbal therapies and naturopathic therapies.
 23. Themethod of claim 1, wherein said matrix is essentially free ofbicarbonate when said matrix is being formed into a gel or a permeablesolid.
 24. The method of claim 1, wherein said matrix is formed fromcomponents, wherein said components are essentially free of bicarbonate.25. The method of claim 1, wherein said measuring of Step F is performedat periodic intervals, and wherein said periodic intervals are set withrespect to one or more of the cells, the tissue and the therapy ortherapies being tested or evaluated.
 26. The method of claim 1, whereinsaid measuring of Step F is performed at periodic intervals for a periodof time of between one hour and 15 days.
 27. The method of claim 25,wherein said measuring is performed at said periodic intervals, and saidintervals are one or more selected from the group consisting of everysix hours, every 12 hours, every 18 hours, every 24 hours, every 36hours, every 48 hours, every 72 hours and every 96 hours.
 28. The methodof claim 25, wherein said periodic intervals are one or more selectedfrom the group consisting of every day, every second day and every thirdday, every fourth day, every fifth, day every sixth day, every seventhday, every eighth day, and every ninth day.
 29. The method of claim 1,wherein said measuring of Step F is performed at non-periodic intervals.30. The method of claim 1, wherein said data obtained by the measuringin Step F is recorded in a fixed media.
 31. The method of claim 30,wherein said fixed media is one or more selected from the groupconsisting of paper, voice recordings, video recordings, photographs,photomicrographs, digital recordings, any fixed digital data means, anycomputer network-facilitated means, infrared recording, ultrasoundrecordings, and magnetic resonance recordings.
 32. The method of claim1, wherein said data is obtained by one or more of a human observer, astill camera, a video camera, an automated still camera, an automatedvideo camera, an infrared camera, and an automated infrared camera. 33.The method of claim 1, wherein said incubating in Step E occurs for aperiod between one hour and 15 days.
 34. The method of claim 1, whereinsaid incubating of said tissue sample portion and said cells occurs fora period of time of at least 1 hour, or at least 10 hours, or at least24 hours, or at least 36 hours or at least 72 hours, or at least 125hours, or at least 175 hours, or at least 200 hours, or at least 250hours or at least 300 hours.
 35. The method of claim 1, wherein saidnutrition comprises one or more fluids, and wherein said one or morefluids are selected from the group consisting of tissue culture medias,concentrated tissue culture medias, and diluted culture medias.
 36. Themethod of claim 35, wherein said concentrated medias are one or moreselected from the group consisting of 10× medias, and any otherconcentrated medias.
 37. The method of claim 1, wherein saidthree-dimensional physiological matrix comprises said nutrition.
 38. Themethod of claim 1, further comprising the step of J) providing at leastone source of additional nutrition to said tissue and said cells so thatsaid parameters can be measured.
 39. The method of claim 38, whereinsaid at least one source of additional nutrition comprises one or moreof tissue culture medias, concentrated tissue culture medias, and anyculture or nutritional medias which are suitable for said tissue sample.40. The method of claim 38, wherein said at least one additional sourceof nutrition is supplemented with one or more selected from the groupconsisting of sera, proteins, sugars, salts, lipids.
 41. The method ofclaim 1, wherein said sufficient nutrition is provided to said tissuesample portions and said cells by placing said nutrition in contact withsaid matrix after said sample has been placed in said matrix and saidmatrix has equilibrated to room temperature, wherein said roomtemperature is in the range of from 10 to 30 degrees Celsius.
 42. Themethod of claim 1, wherein said environment for incubating or culturingsaid cells and said tissue sample portion includes a temperature rangeof from 26 to 43 degrees Celsius, a carbon dioxide tension of from 2.0%to 9.0%, and a humidity range of from 50-100%.
 43. The method of claim1, wherein said matrix comprises a gel, and wherein said matrix isformed in multiple containers such that multiple portions of said samplecan be evaluated, and wherein said matrix is formed in multiplecontainers and said multiple containers comprise a multi-well apparatusor plate.
 44. The method of claim 1, wherein said one or more parameterscomprises one or more of: the distance which individual cells migratefrom said tissue sample, the average distance of migration of a group orpopulation of said cells from said tissue sample, the distance whichindividual cells migrate from said tissue sample with respect to time,the average distance of migration of a group or population of said cellsfrom said tissue sample with respect to time, the velocity of migrationof one or more designated individual said cells per selected timeperiod, the velocity of migration of a group or population of designatedsaid cells per selected time period, the number of migrational cells perunit area of a microscopic visual field, the speed of proliferation ofsaid cells, the speed of unidirectional migration of said cells, thespeed of bi-directional migration of said cells, the speed oftri-directional migration of said cells, the frequency of directionalchange of said migrating cells, the number of directional changes perunit time of said migrating cells, the rate of mitosis of said migratingcells, the number of cells migrating per unit time, the number of cellsin a unit area of a portion of the gel, the change in the number ofcells in a unit area of a portion of the gel with respect to time, thenumber of cells in a unit volume of a portion of the gel with respect totime, and the change in the number of cells in a unit volume of aportion of the gel, the proportion of cells which change migrationaldirection during the test period, the speed of migrating cells in aparticular visual field, the density of migrating cells per unit area ofa visual field, the migrational distance of individual migrating cellsper unit area of a visual field, the average migrational distance of agroup or population of migrating cells per unit area of a visual field,and the number of migrating cells in a particular direction per unitarea of a visual field.
 45. The method of claim 1, wherein saidphysiological matrix has a pH in the range of from 5.0 to 8.0.
 46. Themethod of claim 1, wherein said physiological matrix has a pH in therange of from 7.2 to 7.6.
 47. The method of claim 1 wherein the collagenfibers are selected from the group consisting of type I collagen, typeII collagen, type III collagen and type IV collagen.
 48. A method forevaluating the efficacy of one or more therapies with respect to atleast one tissue from an animal, said tissue comprising cells, and saidtissue being suspected of being abnormal, pre-cancerous or cancerous,said method comprising the steps of A) obtaining a sample of said tissuefrom said animal, wherein said tissue comprises one or more types ofcells, and wherein said sample is divided into portions, and notdissociated by chemical or enzymatic means, wherein said tissuecomprises one or more types of cells, (B) implanting each of said tissuesample portions within a three-dimensional physiological matrix, whereineach of said portions is essentially completely submerged within saidmatrix, said matrix being adapted and arranged for measuring one or moreparameters of the behavior of said tissue portion and said cells,wherein the physiological matrix comprises at least one collagen fibercomponent adapted and arranged such that the cells, if abnormal,cancerous or pre-cancerous, are enabled to grow away from the sampleportion into the matrix, and wherein said collagen fiber componentcomprises at least one natural or synthetic collagen fiber and ispresent in a density of from 1.3 mg/ml to 3.3 mg/ml, C) providingnutrition to said cells of said tissue of said portions, D) subjectingsaid cells and said tissue of said portions to at least one of said oneor more therapies, E) incubating or culturing said tissue and said cellsof said portions in an environment suitable for the growth of said cellsand said tissue for a time sufficient to obtain measurements withrespect to said one or more parameters for each of said portions, and F)measuring, with respect to each of said portions, one or more of saidparameters to obtain data regarding the behaviors of said tissue andsaid cells with respect to said one or more therapies.
 49. A method forevaluating the oncological characteristics of cells from at least onetissue from an animal, said cells or said tissue being suspected ofbeing cancerous or pre-cancerous, said method comprising the steps of A)obtaining a sample of said tissue from said animal, wherein said tissuecomprises one or more types of cells, and wherein said sample is dividedmechanically into portions, and not dissociated by chemical or enzymaticmeans, wherein said tissue comprises one or more types of cells, (B)implanting each of said tissue sample portions within athree-dimensional physiological matrix, wherein each of said portions isessentially completely submerged within said matrix, said matrix beingadapted and arranged for measuring one or more parameters of thebehavior of said tissue portion and said cells, wherein thephysiological matrix comprises at least one collagen fiber componentadapted and arranged such that the cells, if abnormal, cancerous orpre-cancerous, are enabled to grow away from the sample portion into thematrix, and wherein said collagen fiber component comprises at least onenatural or synthetic collagen fiber and is present in a density of from1.3 mg/ml to 3.3 mg/ml, C) providing nutrition to said cells of saidtissue or cells, D) incubating said tissue and said cells in anenvironment suitable for the growth of said cells and said tissue for atime sufficient to obtain measurements with respect to said one or moreparameters, and E) measuring said one or more parameters to obtain dataregarding the behaviors of said tissue and said cells.
 50. The method ofclaim 49, further comprising the step of F) from said data, determiningthe oncological status of said cells.
 51. The method of claim 49,further comprising the step of G) recording said data.
 52. The method ofclaim 49, wherein said implanting of said Step B is effected within 4hours of said Step A.
 53. The method of claim 49, wherein saidimplanting of said Step B is effected within 24 hours of said Step A.54. The method of claim 49, further comprising the step of H)transmitting said data to at least one medical evaluator.
 55. The methodof claim 49, wherein during Step A, said sample quantity is divided intoa plurality of portions, and each of said portions is subjected to saidSteps A through E.
 56. The method of claim 49, wherein said measuring ofStep E is performed more than once with respect to separate portions ofsaid sample, and wherein said measuring is effected during saidincubating of said Step D.
 57. The method of claim 49, wherein saidanimal is one or more selected from the groups consisting of primates,humans, equines, bovines, canids, mammals, felines, canines, murines,porcines, avians, amphibians and reptilians.
 58. The method of claim 50,wherein said evaluator is computerized.
 59. The method of claim 1wherein said tissue is a liquid tissue, a cell suspension or groups ofcells which have been pelletized.
 60. The method of claim 48 whereinsaid tissue is a liquid tissue, a cell suspension or groups of cellswhich have been pelletized.
 61. The method of claim 54, wherein saidevaluator is computerized.
 62. The method of claim 1 wherein thephysiological matrix further comprises fibrin, fibrinogen, extracellularmatrix proteins derived from one or more animals, lamina, fibronectin,or anti-lamina.